Microfluidic rotary flow reactor matrix

ABSTRACT

A microfluidic device comprises a matrix of rotary flow reactors. The microfluidic matrix device offers a solution to the “world-to-chip” interface problem by accomplishing two important goals simultaneously: an economy of scale in reagent consumption is achieved, while simultaneously minimizing pipetting steps. N 2  independent assays can be performed with only 2N+1 pipetting steps, using a single aliquot of enzyme amortized over all reactors. The chip reduces labor relative to conventional fluid handling techniques by using an order of magnitude less pipetting steps, and reduces cost by consuming two to three orders of magnitude less reagents per reaction. A PCR format has immediate applications in medical diagnosis and gene testing. Beyond PCR, the microfluidic matrix chip provides a universal and flexible platform for biological and chemical assays requiring parsimonious use of precious reagents and highly automated processing.

CROSS-REFERENCE TO RELATED APPLICATIONS

The instant nonprovisional patent application claims priority from U.S.provisional patent application No. 60/494,432, filed Aug. 11, 2003. Thispreviously filed provisional patent application is hereby incorporatedby reference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Work described herein has been supported, in part, by DARPA Grant No.DAAD19-00-1-0392 and National Science Foundation Grant No. CTS-0088649.The United States Government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

As described by at least Mitchell in Nat. Biotechnol. 19, 717-721(2001), incorporated by reference herein for all purposes, microfluidictechnology offers many possible benefits in chemistry, biology andmedicine. One important possible benefit is to automate rote work whilereducing the consumption of expensive reagents to the nanoliter orsub-nanoliter scale.

As described in Anal. Chem. 69, 3407-3412 (1997), incorporated byreference herein for all purposes, Hadd et al. developed a microchip forperforming automated enzyme assay, in which precise concentrations ofsubstrate, enzyme, and inhibitor were mixed in nanoliter volumes usingelectrokinetic flow. In Anal. Chem. 74, 2451-2457 (2002), incorporatedby reference herein for all purposes, Fu et al reported an integratedcell sorter with a 1 picoliter minimum active volume by the actuatedvalve.

However, as pointed out by Meldrum et al. in Science 297, 1197-1198(2002), incorporated by reference herein for all purposes, sincemicrofluidic devices must at some point be interfaced to the macroscopicworld, there is a minimum practical volume (of order—1 μl) that can beintroduced into a device. The so-called “world-to-chip” interfaceproblem described by Ramsey in Nat. Biotechnol. 17, 1061-1062 (1999),incorporated by reference herein for all purposes, has plagued themicrofluidic field since its inception. As noted by Ross et al. in Anal.Chem. 74, 2556-2564 (2002), it is always questionable how the desiredeconomies of scale in microfluidics can practically be achieved, unlessan effective approach is developed to solve the mismatch between thosetwo scales.

Although integrated glass capillaries have been used to reduce sampleconsumption for simple titration between two reagents, these devices arefundamentally serial and have the possibility of samplecross-contamination during the loading process. See Farinas et al.,Analytical Biochemistry 295, 138-142 (2001), incorporated by referenceherein for all purposes.

On the other hand, there has been an effort to develop techniques toconcentrate analytes from a large input volume. In Anal. Chem. 73,1627-1633 (2001), incorporated by reference herein for all purposes,Macounova et al. describe microfluidic isoelectric focusing (IEF)techniques. Ross et al. describe temperature gradient focusing (TGF)techniques. However, only a limited species of samples have successfullybeen demonstrated so far.

The challenge associated with realizing the desired economies of scalein microfluidic devices is to simultaneously reduce the number ofpipetting steps needed to load the devices, while amortizing the samplevolume from each pipetting step over a large number of independentassays. As pointed out in U.S. Pat. No. 6,508,988, incorporated byreference herein for all purposes, microfluidic matrix geometries offerthe advantage of performing N² independent reactions with only 2Npipetting steps.

In Lab on a Chip 2, 188-192 (2002), Kikutani et al. used N=2 matricesfor chemical synthesis. In Anal. Chem., 73, 5207-5213 (2001), Ismagilovet al. used N=5 passive matrices to demonstrate two-componentbiochemical assays such as optical detection of enzymatic activity.However, in those devices the reagent consumption scaled only with N,which was so small that there were little practical savings.

Passive devices also have technical limitations in sample metering anddevice operation. For example, precise pressure balancing was requiredduring operation of the devices of Kikutani et al., and the kinetics ofmixing were limited due to the static nature in the devices of Ismagilovet al.

Accordingly, there is a need in the art for microfluidic techniques andapparatuses for addressing the “world-to-chip” interface problem.

SUMMARY OF THE INVENTION

An embodiment of a microfluidic matrix device in accordance with thepresent invention offers an effective solution to the “world-to-chip”interface problem by accomplishing two important goals simultaneously:an economy of scale in reagent consumption is achieved, whilesimultaneously minimizing pipetting steps. N² independent assays can beperformed with only 2N+1 pipetting steps, using a single aliquot of theenzyme that can be amortized over all the reactors. Thus, the chipreduces labor relative to conventional fluid handling techniques byusing an order of magnitude less pipetting steps and reduces cost byconsuming two to three orders of magnitude less reagents per reaction.The demonstrated PCR format has immediate applications in medicaldiagnosis and gene testing. Beyond PCR, the microfluidic matrix chipprovides a universal and flexible platform for biological and chemicalassays that require parsimonious use of precious reagents and highlyautomated processing.

An embodiment of a microfluidic device in accordance with the presentinvention, comprises, a plurality of flow channels defined within anelastomer layer to form a matrix of rotary flow reactors. A first set ofcontrol lines are proximate to and separated from the flow channels byfirst elastomer membranes, the first elastomer membranes actuable tointroduce fluids into the rotary flow reactors and to isolate the rotaryflow reactors. A second set of control lines are proximate to andseparated from the flow channels by second elastomer membranes, thesecond elastomer membranes actuable to cause peristaltic pumping of thefluids within the isolated rotary flow reactors.

A method of conducting a chemical reaction, the method comprising,providing a microfluidic device comprising a plurality of flow channelsdefined within an elastomer layer to form a matrix of rotary flowreactors, a first set of control lines proximate to and separated fromthe flow channels by first elastomer membranes, and a second set ofcontrol lines proximate to and separated from the flow channels bysecond elastomer membranes. First and second chemicals are introduced tothe rotary flow reactors. Pressure is applied to the first set ofcontrol lines to actuate the first elastomer membranes to isolate therotary flow reactors, and pressure is applied to the second set ofcontrol lines to actuate the second elastomer membranes to causeperistaltic pumping of the fluids within the isolated rotary flowreactors.

These and other embodiments of the present invention, as well as itsadvantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIG. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C-7G are illustrations showing steps of a method for forming anelastomeric structure having a membrane formed from a separateelastomeric layer.

FIG. 7H is a front sectional view showing the valve of FIG. 7B in anactuated state.

FIGS. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

FIG. 10 is a front sectional view of the valve of FIG. 7B showingactuation of the membrane.

FIG. 11 is a front sectional view of an alternative embodiment of avalve having a flow channel with a curved upper surface.

FIG. 12A is a top schematic view of an on/off valve.

FIG. 12B is a sectional elevation view along line 23B-23B in FIG. 12A

FIG. 13A is a top schematic view of a peristaltic pumping system.

FIG. 13B is a sectional elevation view along line 24B-24B in FIG. 13A

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.13.

FIG. 15A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 15B is a sectional elevation view along line 26B-26B in FIG. 15A

FIG. 16 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIGS. 17A-D show plan views of one embodiment of a switchable flowarray.

FIGS. 18A-D show plan views of one embodiment of a cell pen arraystructure.

FIGS. 19A-19B show plan and cross-sectional views illustrating operationof one embodiment of a cell cage structure in accordance with thepresent invention.

FIGS. 20A-20D show plan views of operation of a structure utilizingcross-channel injection in accordance with the embodiment of the presentinvention.

FIG. 21 shows a plan view of one embodiment of a rotary mixing structurein accordance with the present invention.

FIG. 22A shows a simplified plan view illustrating a binary treemicrofluidic multiplexor operational diagram.

FIG. 22B shows a simplified plan view illustrating a tertiary treemicrofluidic multiplexor operational diagram.

FIG. 22C shows a simplified cross-sectional view of the generalmicrofluidic architecture of the devices of FIGS. 22A-B.

FIG. 23 plots the number of control lines versus the number of flowlines being controlled, for multiplexors of various base numbers.

FIG. 24 shows a simplified plan view of an embodiment of a microfluidicstructure utilizing control channels to control other control channels.

FIG. 24A shows a simplified cross-sectional view of the structure ofFIG. 24 taken along the line A-A′

FIG. 24B shows a simplified cross-sectional view of the structure ofFIG. 24 taken along the line B-B′.

FIG. 25 shows a simplified cross-sectional view of the generalmicrofluidic architecture of the device of FIGS. 24-24B.

FIG. 26 shows a simplified plan view of an alternative embodiment of amicrofluidic structure utilizing control channels to control othercontrol channels.

FIG. 26A shows a simplified cross-sectional view of the structure ofFIG. 26 taken along the line A-A′.

FIG. 26B shows a simplified cross-sectional view of the structure ofFIG. 26 taken along the line B-B′.

FIG. 27A shows an optical micrograph of a microfluidic comparator chip.

FIG. 27B is a simplified schematic view of the microfluidic comparatorchip of FIG. 27A.

FIGS. 27C-H are enlarged simplified plan views showing loading of thechamber of the microfluidic structure of FIG. 27A.

FIG. 28A shows a simplified schematic plan view of one embodiment of amicrofluidic matrix architecture in accordance with the presentinvention.

FIG. 28B shows an enlarged plan view of a single reactor x.

FIG. 28C shows a simplified cross-sectional view of the microfluidicmatrix of FIG. 28B taken along line 28C-28C′.

FIG. 28D shows a simplified cross-sectional view of the microfluidicmatrix of FIG. 28D taken along line 28D-28D′.

FIGS. 28E-H show enlarged and simplified plan views illustratingsuccessive steps of operation of the microfluidic matrix of FIGS. 28A-D.

FIGS. 29A-D show a set of optical micrographs illustrating the isolationand mixing of different dye solutions by an alternative embodiment of amicrofluidic matrix device in accordance with the present invention.

FIG. 30 shows a two-color image of fluorescent emission from a 20×20matrix chip.

FIG. 31 shows a scanned fluorescent image illustrating use of the matrixchip to explore combinations of forward and reverse primers.

FIG. 32 plots fluorescent ratio vs. the initial concentration of thecDNA templates.

FIG. 33 shows Agarose gel electrophoresis of the PCR amplicon by thechip or by a microtube.

FIG. 34 shows an exploded view of an alternative embodiment of amicrofluidic structure for performing PCR.

FIG. 35 plots fluorescence versus time for nucleic acids amplified byPCR utilizing the microfluidic structure shown in FIG. 34.

FIGS. 36A-D are simplified plan views showing operation of analternative embodiment of a microfluidic matrix architecture inaccordance with the present invention.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (2d ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE ANDTECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, THEHARPER COLLINS DICTIONARY OF BIOLOGY (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

A “flow channel” refers generally to a flow path through which asolution can flow.

The term “valve” unless otherwise indicted refers to a configuration inwhich a flow channel and a control channel intersect and are separatedby an elastomeric membrane that can be deflected into or retracted fromthe flow channel in response to an actuation force.

The term “elastomer” and “elastomeric” has its general meaning as usedin the art. Thus, for example, Allcock et al. (Contemporary PolymerChemistry, 2nd Ed.) describes elastomers in general as polymers existingat a temperature between their glass transition temperature andliquefaction temperature. Elastomeric materials exhibit elasticproperties because the polymer chains readily undergo torsional motionto permit uncoiling of the backbone chains in response to a force, withthe backbone chains recoiling to assume the prior shape in the absenceof the force. In general, elastomers deform when force is applied, butthen return to their original shape when the force is removed. Theelasticity exhibited by elastomeric materials can be characterized by aYoung's modulus. The elastomeric materials utilized in the microfluidicdevices disclosed herein typically have a Young's modulus of betweenabout 1 Pa-1 TPa, in other instances between about 10 Pa-100 GPa, instill other instances between about 20 Pa-1 GPa, in yet other instancesbetween about 50 Pa-10 MPa, and in certain instances between about 100Pa-1 MPa. Elastomeric materials having a Young's modulus outside ofthese ranges can also be utilized depending upon the needs of aparticular application.

Some of the microfluidic devices described herein are fabricated from anelastomeric polymer such as GE RTV 615 (formulation), a vinyl-silanecrosslinked (type) silicone elastomer (family). However, the presentmicrofluidic systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. Given the tremendous diversity of polymer chemistries,precursors, synthetic methods, reaction conditions, and potentialadditives, there are a large number of possible elastomer systems thatcan be used to make monolithic elastomeric microvalves and pumps. Thechoice of materials typically depends upon the particular materialproperties (e.g., solvent resistance, stiffness, gas permeability,and/or temperature stability) required for the application beingconducted. Additional details regarding the type of elastomericmaterials that can be used in the manufacture of the components of themicrofluidic devices disclosed herein are set forth in U.S. applicationSer. No. 09/605,520, and PCT Application No. 00/17740, both of which areincorporated herein by reference in their entirety.

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused herein to include a polymeric form of nucleotides of any length,including, but not limited to, ribonucleotides or deoxyribonucleotides.There is no intended distinction in length between these terms. Further,these terms refer only to the primary structure of the molecule. Thus,in certain embodiments these terms can include triple-, double- andsingle-stranded DNA, as well as triple-, double- and single-strandedRNA. They also include modifications, such as by methylation and/or bycapping, and unmodified forms of the polynucleotide. More particularly,the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide,”include polydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), any other type ofpolynucleotide which is an N- or C-glycoside of a purine or pyrimidinebase, and other polymers containing nonnucleotidic backbones, forexample, polyamide (e.g., peptide nucleic acids (PNAs)) andpolymorpholino (commercially available from the Anti-Virals, Inc.,Corvallis, Oreg., as Neugene) polymers, and other syntheticsequence-specific nucleic acid polymers providing that the polymerscontain nucleobases in a configuration which allows for base pairing andbase stacking, such as is found in DNA and RNA.

A “probe” is an nucleic acid capable of binding to a target nucleic acidof complementary sequence through one or more types of chemical bonds,usually through complementary base pairing, usually through hydrogenbond formation, thus forming a duplex structure. The probe binds orhybridizes to a “probe binding site.” The probe can be labeled with adetectable label to permit facile detection of the probe, particularlyonce the probe has hybridized to its complementary target. The labelattached to the probe can include any of a variety of different labelsknown in the art that can be detected by chemical or physical means, forexample. Suitable labels that can be attached to probes include, but arenot limited to, radioisotopes, fluorophores, chromophores, mass labels,electron dense particles, magnetic particles, spin labels, moleculesthat emit chemiluminescence, electrochemically active molecules,enzymes, cofactors, and enzyme substrates. Probes can vary significantlyin size. Some probes are relatively short. Generally, probes are atleast 7 to 15 nucleotides in length. Other probes are at least 20, 30 or40 nucleotides long. Still other probes are somewhat longer, being atleast 50, 60, 70, 80, 90 nucleotides long. Yet other probes are longerstill, and are at least 100, 150, 200 or more nucleotides long. Probescan be of any specific length that falls within the foregoing ranges aswell.

A “primer” is a single-stranded polynucleotide capable of acting as apoint of initiation of template-directed DNA synthesis under appropriateconditions (i.e., in the presence of four different nucleosidetriphosphates and an agent for polymerization, such as, DNA or RNApolymerase or reverse transcriptase) in an appropriate buffer and at asuitable temperature. The appropriate length of a primer depends on theintended use of the primer but typically is at least 7 nucleotides longand, more typically range from 10 to 30 nucleotides in length. Otherprimers can be somewhat longer such as 30 to 50 nucleotides long. Shortprimer molecules generally require cooler temperatures to formsufficiently stable hybrid complexes with the template. A primer neednot reflect the exact sequence of the template but must be sufficientlycomplementary to hybridize with a template. The term “primer site” or“primer binding site” refers to the segment of the target DNA to which aprimer hybridizes. The term “primer pair” means a set of primersincluding a 5′ “upstream primer” that hybridizes with the complement ofthe 5′ end of the DNA sequence to be amplified and a 3′ “downstreamprimer” that hybridizes with the 3′ end of the sequence to be amplified.

A primer that is “perfectly complementary” has a sequence fullycomplementary across the entire length of the primer and has nomismatches. The primer is typically perfectly complementary to a portion(subsequence) of a target sequence. A “mismatch” refers to a site atwhich the nucleotide in the primer and the nucleotide in the targetnucleic acid with which it is aligned are not complementary. The term“substantially complementary” when used in reference to a primer meansthat a primer is not perfectly complementary to its target sequence;instead, the primer is only sufficiently complementary to hybridizeselectively to its respective strand at the desired primer-binding site.

The term “complementary” means that one nucleic acid is identical to, orhybridizes selectively to, another nucleic acid molecule. Selectivity ofhybridization exists when hybridization occurs that is more selectivethan total lack of specificity. Typically, selective hybridization willoccur when there is at least about 55% identity over a stretch of atleast 14-25 nucleotides, preferably at least 65%, more preferably atleast 75%, and most preferably at least 90%. Preferably, one nucleicacid hybridizes specifically to the other nucleic acid. See M. Kanehisa,Nucleic Acids Res. 12:203 (1984).

The term “label” refers to a molecule or an aspect of a molecule thatcan be detected by physical, chemical, electromagnetic and other relatedanalytical techniques. Examples of detectable labels that can beutilized include, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, enzymes linked to nucleic acidprobes and enzyme substrates. The term “detectably labeled” means thatan agent has been conjugated with a label or that an agent has someinherent characteristic (e.g., size, shape or color) that allows it tobe detected without having to be conjugated to a separate label.

I. Microfabrication Overview

The following discussion relates to formation of microfabricated fluidicdevices utilizing elastomer materials, as described generally in U.S.patent application Ser. Nos. 10/118,466, filed Apr. 5, 2002, Ser. No.10/265,473 filed Oct. 4, 2002, Ser. No. 10/118,466 filed Apr. 5, 2002,Ser. No. 09/826,585 filed Apr. 6, 2001, Ser. No. 09/724,784 filed Nov.28, 2000, and Ser. No. 09/605,520, filed Jun. 27, 2000. Thesepreviously-filed patent applications are hereby incorporated byreference for all purposes.

1. Methods of Fabricating

Exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded. In analternative method, each layer of elastomer may be cured “in place”. Inthe following description “channel” refers to a recess in theelastomeric structure which can contain a flow of fluid or gas.

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending therealong. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIGS. 7A and 7B, when elastomericstructure 24 has been sealed at its bottom surface to planar substrate14, recess 21 will form a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIGS. 7A and 7B, flow channels 30 and 32 arepreferably disposed at an angle to one another with a small membrane 25of substrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-7G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending therealong is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C-7G offers the advantage of permitting the membrane portion tobe composed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

2. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30 and 32 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30 and 32 have widths of about 1 to 1000 microns.A non-exclusive list of other ranges of widths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30 and 32 have depths of about 1 to 100 microns. Anon-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

The Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, elastomeric layer 22 of FIG. 1 is 50 microns toseveral centimeters thick, and more preferably approximately 4 mm thick.A non-exclusive list of ranges of thickness of the elastomer layer inaccordance with other embodiments of the present invention is betweenabout 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

3. Soft Lithographic Bonding

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In a preferred aspect, the various layers of elastomer are boundtogether in a heterogeneous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogeneous aspect, the bonding process usedto bind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from pure acrylated Urethane Ebe270. A thin bottom layer was spin coated at 8000 rpm for 15 seconds at170° C. The top and bottom layers were initially cured under ultravioletlight for 10 minutes under nitrogen utilizing a Model ELC 500 devicemanufactured by Electrolite corporation. The assembled layers were thencured for an additional 30 minutes. Reaction was catalyzed by a 0.5%vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.The resulting elastomeric material exhibited moderate elasticity andadhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr 245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers may beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer will create a bond between the elastomeric layers andcreate a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold10 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 12. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SJR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

4. Suitable Elastomeric Materials

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, the elastomeric layersmay preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, Polybutadiene, Polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Polyisobutylene:

Pure Polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (˜1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thePolyisobutylene backbone, which may then be vulcanized as above.

Poly(styrene-butadiene-styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

5. Operation of Device

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown. Membrane 25 separates the flow channels, forming the top offirst flow channel 30 and the bottom of second flow channel 32. As canbe seen, flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL.

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:w=(BPb ⁴)/(Eh ³), where:  (1)

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and    -   h=plate thickness.

Thus even in this extremely simplified expression, deflection of anelastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force. Becauseeach of these parameters will vary widely depending upon the actualdimensions and physical composition of a particular elastomeric devicein accordance with the present invention, a wide range of membranethicknesses and elasticity's, channel widths, and actuation forces arecontemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 8A and 8B illustrate valve opening vs. applied pressure for a 100μm wide first flow channel 30 and a 50 μm wide second flow channel 32.The membrane of this device was formed by a layer of General ElectricSilicones RTV 615 having a thickness of approximately 30 μm and aYoung's modulus of approximately 750 kPa. FIGS. 21 a and 21 b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.

For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebrospinal fluid, pressure present in the intra-ocularspace, and the pressure exerted by muscles during normal flexure. Othermethods of regulating external pressure are also contemplated, such asminiature valves, pumps, macroscopic peristaltic pumps, pinch valves,and other types of fluid regulating equipment such as is known in theart.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

FIG. 9 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 9. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force (≦40 kPa). Thus, τclose is expected to be smallerthan τopen. There is also a lag between the control signal and controlpressure response, due to the limitations of the miniature valve used tocontrol the pressure. Calling such lags t and the 1/e time constants τ,the values are: topen=3.63 ms, τopen=1.88 ms, tclose=2.15 ms,τclose=0.51 ms. If 3τ each are allowed for opening and closing, thevalve runs comfortably at 75 Hz when filled with aqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C-7H.)

When experimentally measuring the valve properties as illustrated inFIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH≧8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

6. Flow Channel Cross Sections

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 10, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 10, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 10, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 11, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 10.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

7. Networked Systems

FIGS. 12A and 12B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 13A and 13Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

Referring first to FIGS. 12A and 12B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 12 to 15, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIGS. 13A and 13B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

In experiments performed by the inventors, a pumping rate of 2.35 nL/swas measured by measuring the distance traveled by a column of water inthin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. Coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 13.

FIGS. 15A and 15B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 12. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 16 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing thereabove (for example, membranes25A, 25B, and 25C in FIGS. 15A and 15B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 15A and 15B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 16 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, and 32D. Control channels 32A, 32B, 32C, and 32D are adapted toshut off fluid flows F1, F2, F3, F4, F5 and F6 passing through parallelflow channels 30A, 30B, 30C, 30D, 30E and 30F using any of the valvingsystems described above, with the following modification.

Each of control lines 32A, 32B, 32C, and 32D have both wide and narrowportions. For example, control line 32A is wide in locations disposedover flow channels 30A, 30C and 30E. Similarly, control line 32B is widein locations disposed over flow channels 30B, 30D and 30F, and controlline 32C is wide in locations disposed over flow channels 30A, 30B, 30Eand 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log 2n) controllines.

8. Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 17A to17D. FIG. 17A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 17Bis a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width therealong, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned overtop of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 17C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 17D.

As can be seen in FIG. 17C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 17D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIGS. 17A-D allows a switchable flow array tobe constructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

9. Cell Pen

In yet a further application of the present invention, an elastomericstructure can be utilized to manipulate organisms or other biologicalmaterial. FIGS. 18A-18D show plan views of one embodiment of a cell penstructure in accordance with the present invention.

Cell pen array 4400 features an array of orthogonally-oriented flowchannels 4402, with an enlarged “pen” structure 4404 at the intersectionof alternating flow channels. Valve 4406 is positioned at the entranceand exit of each pen structure 4404. Peristaltic pump structures 4408are positioned on each horizontal flow channel and on the vertical flowchannels lacking a cell pen structure.

Cell pen array 4400 of FIG. 18A has been loaded with cells A-H that havebeen previously sorted. FIGS. 18B-18C show the accessing and removal ofindividually stored cell C by 1) opening valves 4406 on either side ofadjacent pens 4404 a and 4404 b, 2) pumping horizontal flow channel 4402a to displace cells C and G, and then 3) pumping vertical flow channel4402 b to remove cell C. FIG. 18D shows that second cell G is moved backinto its prior position in cell pen array 4400 by reversing thedirection of liquid flow through horizontal flow channel 4402 a. Thecell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access.

While the embodiment shown and described above in connection with FIGS.18A-18D utilizes linked valve pairs on opposite sides of the flowchannel intersections, this is not required by the present invention.Other configurations, including linking of adjacent valves of anintersection, or independent actuation of each valve surrounding anintersection, are possible to provide the desired flow characteristics.With the independent valve actuation approach however, it should berecognized that separate control structures would be utilized for eachvalve, complicating device layout.

10. Cell Cage

The cell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access. However,living organisms such as cells may require a continuous intake of foodsand expulsion of wastes in order to remain viable. Accordingly, FIGS.19A and 19B show plan and cross-sectional views (along line 45B-45B′)respectively, of one embodiment of a cell cage structure in accordancewith the present invention.

Cell cage 4500 is formed as an enlarged portion 4500 a of a flow channel4501 in an elastomeric block 4503 in contact with substrate 4505. Cellcage 4500 is similar to an individual cell pen as described above inFIGS. 18A-18D, except that ends 4500 b and 4500 c of cell cage 4500 donot completely enclose interior region 4500 a. Rather, ends 4500 a and4500 b of cage 4500 are formed by a plurality of retractable pillars4502.

Specifically, control channel 4504 overlies pillars 4502. When thepressure in control channel 4504 is reduced, elastomeric pillars 4502are drawn upward into control channel 4504, thereby opening end 4500 bof cell cage 4500 and permitting a cell to enter. Upon elevation ofpressure in control channel 4504, pillars 4502 relax downward againstsubstrate 4505 and prevent a cell from exiting cage 4500.

Elastomeric pillars 4502 are of a sufficient size and number to preventmovement of a cell out of cage 4500, but also include gaps 4508 whichallow the flow of nutrients into cage interior 4500 a in order tosustain cell(s) stored therein. Pillars 4502 on opposite end 4500 c aresimilarly configured beneath second control channel 4506 to permitopening of the cage and removal of the cell as desired.

11. Cross-Channel Injector

The cross-flow channel architecture illustrated shown in FIGS. 18A-18Dcan be used to perform functions other than the cell pen just described.For example, the cross-flow channel architecture can be utilized inmixing applications.

This is shown in FIGS. 20A-D, which illustrate a plan view of mixingsteps performed by a microfabricated structures in accordance anotherembodiment of the present invention. Specifically, portion 7400 of amicrofabricated mixing structure comprises first flow channel 7402orthogonal to and intersecting with second flow channel 7404. Controlchannels 7406 overlie flow channels 7402 and 7404 and form valve pairs7408 a-b and 7408 c-d that surround each intersection 7412.

As shown in FIG. 20A, valve pair 7408 c-d is initially opened whilevalve pair 7408 a-b is closed, and fluid sample 7410 is flowed tointersection 7412 through flow channel 7404. Valve pair 7408 a-b is thenactuated, trapping fluid sample 7410 at intersection 7412.

Next, as shown in FIG. 20B, valve pairs 7408 c-d are closed and 7408 a-bare opened, such that fluid sample 7410 is injected from intersection7412 into flow channel 7402 bearing a cross-flow of fluid. The processshown in FIGS. 20A-B can be repeated to accurately dispense any numberof fluid samples down cross-flow channel 7402.

While the embodiment of a process-channel flow injector structure shownin FIGS. 20A-B feature channels intersecting at a single junction, thisis not required by the present invention. Thus FIG. 20C shows asimplified plan view of another embodiment of an injection structure inaccordance with the present invention, wherein junction 7450 betweenintersecting flow channels 7452 is extended to provide additional volumecapacity. FIG. 20D shows a simplified plan view of yet anotherembodiment of an injection structure in accordance with the presentinvention, wherein elongated junction 7460 between intersecting flowchannels 7462 includes branches 7464 to provide still more injectionvolume capacity.

12. Rotary Mixing Structure

Microfluidic control and flow channels in accordance with embodiments ofthe present invention may be oriented to rotary pump design whichcirculates fluid through a closed circuit flow channel. As used hereinthe term “closed circuit” has the meaning known in the art and refers toconfigurations that are circular and variations thereof such asellipsoids and ovals, as well as flow circuit paths having corners asare created by triangular, rectangular, or more complex shapes.

As illustrated in FIG. 21, a layer with flow channels 2100 has aplurality of sample inputs 2102, a mixing T-junction 2104, a centralcirculation loop 2106 (i.e., the substantially circular flow channel),and an output channel 2108. The overlay of control channels with a flowchannel can form a microvalve. This is so because the control and flowchannels are separated by a thin elastomeric membrane that can bedeflected into the flow channel or retracted therefrom.

The substantially circular central loop and the control channels thatintersect with it form the central part of the rotary pump. The pump(s)which cause solution to be flowed through the substantially circularflow channel consist of a set of at least three control channels 2110a-c that are adjacent to one another and which intersect thesubstantially circular branch flow channel 2106 (i.e., the centralloop).

When a series of on/off actuation sequences, such a 001, 011, 010, 110,100, 101, are applied to the control channels, the fluid in the centralloop can be peristaltically pumped in a chosen direction, eitherclockwise or counterclockwise. The peristaltic pumping action resultsfrom the sequential deflection of the membranes separating the controlchannels and flow channel into or out of the flow channel.

In general, the higher the actuation frequency, the faster the fluidrotates through the central loop. However, a point of saturation mayeventually be reached at which increased frequency does not result infaster fluid flow. This is primarily due to limitations in the rate atwhich the membrane can return to an unactuated position.

While the system shown in FIG. 21 shows each pump including threecontrol channels, a different number of control channels can beutilized, for example, a single serpentine control channel havingmultiple cross-over points could be used.

A variety of different auxiliary flow channels which are in fluidcommunication with the central loop can be utilized to introduce andwithdrawn sample and reactant solutions from the central loop.Similarly, one or more exit or outlet flow channels in fluidcommunication with the central loop can be utilized to remove solutionfrom the central loop. For example, control valves can be utilized atthe inlet(s) and the outlet(s) to prevent solution flow into or out fromthe central loop.

Flow channel sizes and shapes can vary. With certain devices, thediameter of the channel tends to range from about 1 mm to 2 cm, althoughthe diameter can be considerably larger in certain devices (e.g., 4, 6,8, or 10 cm). Limits on how small the diameter of the circular flowchannel can be are primarily a function of the limits imposed by themultilayer soft lithography processes. Channel widths (either flow orcontrol) usually vary between 30 μm and 250 μm. However, channel widthin some devices is as narrow as 1 um. Channels of larger widths can alsobe utilized, but generally require some type of structural supportwithin the flow channel. Channel height generally varies between 5 and50 μm. In flow channels having a width of 100 μm or less, the channelheight may be 1 μm or smaller. The flow channel is typically rounded toallow for complete blockage of the channel once the membrane isdeflected into the channel. In some devices, the channels have shapessuch as octagons or hexagons. In certain devices, the flow channels arerounded and 100 μm wide and 10 μm high and control channels are 100 μmwide and 10 μm high. One system that has been utilized in certainstudies has utilized a central loop having a diameter of 2 cm, a flowchannel width of 100 μm and a depth of 10 μm.

While the channels typically have the foregoing sizes and shapes, itshould be recognized that the devices provided herein are not limited tothese particular sizes and shapes. For example, branches present in aclosed circuit flow channel may serve to control the dispersion andhence mixing of materials flowed therein.

13. Microfluidic Large-Scale Integration

The previous section has described monolithic microvalves that aresubstantially leakproof and scalable, and has also described methods forfabricating these microvalves. For the relatively simple assemblies ofmicrofluidic valves previously described, each fluid flow channel may becontrolled by its own individual valve control channel. However, such anon-integrated control strategy cannot be practicably implemented formore complex assemblies comprising thousands or even tens of thousandsof individually addressable valves. Accordingly, a variety of techniquesmay be applied alone or in combination to allow for the fabrication oflarge scale integrated microfluidic devices having individuallyaddressable valves.

Techniques useful for implementing large scale integrated microfluidicstructures in accordance with embodiments of the present invention arediscussed in detail in pending U.S. nonprovisional patent applicationNo. 10/670,997. One technique allowing for the fabrication of largescale int microfluidic devices is the use of multiplexor structures.

The use of multiplexor structures has previously been described inconnection with a single set of control lines overlying a single set offlow channels. FIG. 22A shows a simplified plan view illustrating amicrofluidic binary tree multiplexor operational diagram. Flow channels1900 defined in a lower elastomer layer contain the fluid of interest,while control channels 1902 defined in an overlying elastomer layerrepresent control lines containing an actuation fluid such as air orwater. Valves 1904 are defined by the membranes formed at theintersection of the wider portion 1902 a of a control channel 1902 witha flow channel 1900. The actuation pressure is chosen so that only thewide membranes are fully deflected into the flow channel 1900.Specifically, the multiplexor structure is based on the sharp increasein pressure required to actuate a valve as the ratio of control channelwidth: flow channel width is decreased.

The multiplexor structure shown in FIG. 22A is in the form of a binarytree of valves where each stage selects one out of two total groups offlow channels. In the multiplexor embodiment shown in FIG. 22A, eachcombination of open/closed valves in the multiplexor selects for asingle channel, so that n flow channels can be addressed with only 2log₂ n control channels.

By using multiplexed valve systems, the power of the binary systembecomes evident: only about 20 control channels are required tospecifically address 1024 flow channels. This allows a large number ofelastomeric microvalves to perform complex fluidic manipulations withinthese devices, while the interface between the device and the externalenvironment is simple and robust.

FIG. 22B shows a simplified plan view of an alternative embodiment of amultiplexor structure in accordance with the present invention.Multiplexor structure 1950 comprises control channels 1952 formed in anelastomer layer overlying flow channels 1954 of an underlying elastomerlayer. Operating under the same physical principles of the multiplexorof FIG. 22A, multiplexor 1950 comprises a tertiary tree of valves, whereeach stage comprises three bits (“a trit”) and selects one out of threetotal groups of flow channels. Each combination of open/closed valves inthe multiplexor 1950 selects for a single channel, so that n flowchannels can be addressed with only 3 log₃ n control channels.

The general microfluidic flow architecture of either of the basicmultiplexor devices shown in FIGS. 22A-B may be generically representedin the simplified cross-sectional view of FIG. 22C, wherein secondelastomer layer E2 defining control channel network C overlies firstelastomer layer E1 defining flow channel network F.

The base 3 multiplexor of FIG. 22B is the most efficient design that maybe used to address large numbers of “flow” channels. This is because thex log_(x) n valve is minimized where e is used for the base of the log.As fractions are not used for the base of an actual multiplexor, themost efficient multiplexor structure is achieved where the value of x=3,the integer closest to e (˜2.71828).

To highlight this point, TABLE 2 compares the efficiency of the base 2multiplexor with the base 3 multiplexor.

TABLE 2 Number of Flow Lines Controlled by Control Lines EnhancedEfficiency Number of Base 2 Base 3 of Base 3 Multiplexor Control LinesMultiplexor Multiplexor Structure 6 8 9 +1 9 23 27 +4 12 64 81 +17 15181 243 +62 18 512 729 +217

While the above description has focused upon various multiplexorstructures utilizing stages having the same base number, this is notrequired by the present invention. Alternative embodiments ofmultiplexor structures in accordance with the present invention maycomprise stages of unlike base numbers. For example, a two-stagemultiplexor consisting of a bit stage and a trit stage represents themost efficient way of addressing six flow channels. The order of thestages is arbitrary, and will always result in the same number of flowlines being controlled. The use of multiplexor structures comprisingdifferent binary and tertiary stages allows the efficient addressing ofany number of “flow” channels that are the product of the numbers 2 and3.

A multiplexor may conceivably use any base number. For example, five mayalso be used as the base number, if necessary. However, efficiency inutilization of control lines diminishes as the number of control linesmoves away from the value of e. This is shown in FIG. 23, which plotsthe number of control lines versus the number of flow lines beingcontrolled, for multiplexor structures having different base numbers.

Another technique allowing for the fabrication of large scale integrated(LSI) microfluidic devices is the use of multiple layers of controllines. FIGS. 24-24B illustrate this approach. FIG. 24 shows a plan viewof one embodiment of a microfluidic device having a first control linecontrolled by a second control line. FIG. 24A shows a cross-sectionalview of the microfluidic device of FIG. 24, taken along line A-A′. FIG.24B shows a cross-sectional view of the microfluidic device of FIG. 24,taken along line B-B′.

Microfluidic structure 2100 comprises two flow channels 2102 a-b formedin lowermost elastomer layer 2104. First control channel network 2106including first inlet 2106 a in fluid communication with first andsecond branches 2106 b and 2106 c, is formed in a second elastomer layer2108 overlying first elastomer layer 2104. First branch 2106 b of firstcontrol channel network 2106 includes widened portion 2110 overlyingfirst flow channel 2102 a to define first valve 2112. Second branch 2106c of first control channel network 2106 includes widened portion 2114overlying second flow channel 2102 b to define second valve 2116.

Second control channel network 2118 comprising third control channel2118 a is formed in third elastomer layer 2120 overlying secondelastomer layer 2108. Third control channel 2118 a includes widenedportion 2118 b overlying first branch 2106 b of first control channelnetwork 2106 to form valve 2122.

The microfluidic device illustrated in FIGS. 24-24B may be operated asfollows. A fluid that is to be manipulated is present in flow channels2102 a and 2102 b. Application of a pressure to the first controlchannel network 2106 causes the membranes of valves 2112 and 2116 todeflect downward into their respective flow channels 2102 a and 2102 b,thereby valving flow through the flow channels.

Application of a pressure to second control channel network 2118 causesthe membrane of valve 2122 to deflect downward into underlying firstbranch 2106 c only of first control channel network 2106. This fixes thevalve 2112 in its deflected state, in turn allowing the pressure withinthe first control channel network 2106 to be varied without affectingthe state of valve 2112.

The general architecture of the microfluidic device depicted in FIGS.24-24B is summarized in the simplified cross-sectional view of FIG. 25.Specifically, elastomeric device 2200 comprises lowest elastomer layerE1 defining flow channel network F, underlying second elastomer layer E2defining first control channel network C1. First control channel networkC1 in turn underlies second control channel network C2 that is definedwithin third elastomer layer E3.

While the embodiment of the microfluidic device of FIGS. 24-24B isdescribed as being fabricated from three separate elastomer layers, thisis not required by the present invention. Large scale integratedmicrofluidic structures in accordance with embodiments of the presentinvention featuring multiplexed control lines may be fabricatedutilizing only two elastomer layers. This approach is shown andillustrated in connection with FIGS. 26-26B.

FIG. 26 shows a simplified plan view of a microfabricated elastomerdevice including first and second flow channels 2300 a and 2300 b, andfirst branched control channel network 2302 overlying flow channels 2300a and 2300 b to define valves 2304 and 2306 respectively. FIG. 26A showsa cross-sectional view of the microfabricated elastomer device of FIG.26, taken along line A-A′, with flow channel 2300 a defined in lowerelastomer layer 2308, and first control channel 2302 defined in upperelastomer layer 2310.

Lower elastomer layer 2308 further comprises a second control channelnetwork 2312 running underneath first control channel 2302 to definevalve 2314. Accordingly, FIG. 26B shows a cross-sectional view of themicrofabricated elastomer device of FIG. 26, taken along line B-B′.While present in the same (lower) elastomer layer 2308, flow channelnetwork 2300 and second control channel network 2312 are separate and donot intersect one another.

As represented in the simplified cross-sectional view of FIG. 27,separate flow channel network F and control channel network C2 may thusbe present on a single (lower) elastomer layer E1 that is overlaid byanother elastomer layer E2 defining only a control channel network C1.

The microfluidic device illustrated in FIGS. 26-26B may be operated asfollows. A fluid that is to be manipulated is present in flow channels2300 a and 2300 b. Application of a pressure to the first controlchannel network 2302 causes the membranes of valves 2304 to deflectdownward into their respective flow channels 2300 a and 2300 b, therebyvalving flow through the flow channels.

Application of a pressure to second control channel network 2312 causesthe membrane of valve 2314 to deflect upward into the overlying branchof first control channel network 2302. This fixes the valve 2314 in itsdeflected state, in turn allowing the pressure within the first controlnetwork 2302 to be varied without affecting the state of valve 2314. Incontrast with the embodiment shown in FIG. 24, the microfluidic deviceof FIGS. 26-26B features a valve that operates by deflecting upward intoan adjacent control channel in response to an elevated pressure.

FIG. 27A shows an optical micrograph of a microfluidic comparator chip3000 microfabricated with large scale integration technology which isanalogous to an array of 256 comparators. Specifically, a second devicecontaining 2056 microvalves was designed which is capable of performingmore complex fluidic manipulations. The various inputs have been loadedwith colored food dyes to visualize the channels and sub-elements of thefluidic logic. FIG. 27B shows a simplified schematic plan view of oneportion of the chip of FIG. 27A.

Comparator chip 3000 is formed from a pair of parallel, serpentine flowchannels 3002 and 3004 having inlets 3002 a and 3004 a respectively, andhaving outlets 3002 b and 3004 b respectively, that are intersected atvarious points by branched horizontal rows of flow channels 3006.Portions of the horizontal flow channels located between the serpentineflow channels define mixing locations 3010.

A first barrier control line 3012 overlying the center of the connectingchannels is actuable to create adjacent chambers, and is deactivable toallow the contents of the adjacent chambers to mix. A second barriercontrol line 3014 doubles back over either end of the adjacent chambersto isolate them from the rest of the horizontal flow channels.

One end 3006 a of the connecting horizontal flow channel 3006 is influid communication with pressure source 3016, and the other end 3006 bof the connecting horizontal flow channel 3006 is in fluid communicationwith a sample collection output 3018 through multiplexor 3020.

FIGS. 27C-H show simplified enlarged plan views of operation of onemixing element of the structure of FIGS. 27A-B. FIG. 27C shows themixing element prior to loading, with the mixer barrier control line andwrap-around barrier control line unpressurized. FIG. 27D showspressurization of the wrap-around barrier control line and barrier mixerline to activate isolation valves and separation valve to defineadjacent chambers 3050 and 3052. FIG. 27E shows loading of the chamberswith a first component and a second component by flowing these materialsdown the respective flow channels. FIG. 27F shows pressurization of thevertical compartmentalization control line 3025 and the isolation todefine the adjacent chambers.

FIG. 27G shows depressurization of the mixing barrier control channel todeactivate the separation barrier valve, thereby allowing the differentcomponents present in the adjacent chambers to mix freely.

FIG. 27H shows the deactivation of barrier the isolation control line,causing deactivation of the isolation valves, followed by application ofpressure to the control line and deactivation of the multiplexor toallow the combined mixture to be recovered.

In the case of the device shown in FIGS. 27A-H, two different reagentscan be separately loaded, mixed pair wise, and selectively recovered,making it possible to perform distinct assays in 256 sub-nanoliterreaction chambers and then recover a particularly interesting reagent.The microchannel layout consists of four central columns in the flowlayer consisting of 64 chambers per column, with each chamber containing˜750 pL of liquid after compartmentalization and mixing. Liquid isloaded into these columns through two separate inputs under low externalpressure (˜20 kPa), filling up the array in a serpentine fashion.Barrier valves on the control layer function to isolate the samplefluids from each other and from channel networks on the flow layer usedto recover the contents of each individual chamber. These networksfunction under the control of a multiplexor and several other controlvalves.

The storage array and comparator microfluidic devices shown in FIGS.27A-H was fabricated with multilayer soft lithography techniques usingtwo distinct layers. The “control” layer, which harbors all channelsrequired to actuate the valves, is situated on top of the “flow” layer,which contains the network of channels being controlled. A valve iscreated where a control channel crosses a flow channel. The resultingthin membrane in the junction between the two channels can be deflectedby hydraulic or pneumatic actuation. All biological assays and fluidmanipulations are performed on the “flow” layer.

Master molds for the microfluidic channels were made by spin-coatingpositive photoresist (Shipley SJR 5740) on silicon 9 μm high andpatterning them with high resolution (3386 dpi) transparency masks. Thechannels on the photoresist molds were rounded at 120° C. for 20 minutesto create a geometry that allows full valve closure.

The devices were fabricated by bonding together two layers of two-partcure silicone (Dow Corning Sylgard 184) cast from the photoresist molds.The bottom layer of the device, containing the “flow” channels, isspin-coated with 20:1 part A:B Sylgard at 2500 rpm for 1 minute. Theresulting silicone layer is ˜30 μm thick. The top layer of the device,containing the “control” channels, is cast as a thick layer (˜0.5 cmthick) using 5:1 part A:B Sylgard using a separate mold. The two layersare initially cured for 30 minutes at 80° C.

Control channel interconnect holes are then punched through the thicklayer (released from the mold), after which it is sealed, channel sidedown, on the thin layer, aligning the respective channel networks.Bonding between the assembled layers is accomplished by curing thedevices for an additional 45-60 minutes at 80° C. The resultingmultilayer devices are cut to size and mounted on RCA cleaned No. 1, 25mm square glass coverslips, or onto coverslips spin coated with 5:1 partA:B Sylgard at 5000 rpm and cured at 80° C. for 30 minutes, followed byincubation at 80° C. overnight.

Simultaneous addressing of multiple non-contiguous flow channels isaccomplished by fabricating control channels of varying width whilekeeping the dimension of the flow channel fixed (100 μm wide and 9 μmhigh). The pneumatic pressure in the control channels required to closethe flow channels scales with the width of the control channel, makingit simple to actuate 100 μm×100 μm valves at relatively low pressures(˜40 kPa) without closing off the 50 μm×100 μm crossover regions, whichhave a higher actuation threshold.

Introduction of fluid into these devices is accomplished through steelpins inserted into holes punched through the silicone. Unlikemicromachined devices made out of hard materials with a high Young'smodulus, silicone is soft and forms a tight seal around the input pins,readily accepting pressures of up to 300 kPa without leakage.Computer-controlled external solenoid valves allow actuation ofmultiplexors, which in turn allow complex addressing of a large numberof microvalves.

II. Microfluidic Matrix Architecture

FIG. 28A shows a simplified schematic plan view of one embodiment of amicrofluidic matrix architecture 6000 in accordance with the presentinvention, showing the N=20 matrix chip layout, and the various input,output and control ports. The scale bar of FIG. 28A is 6.4 mm. FIG. 28Bshows an enlarged plan view of one reactor from the upper left handcorner of the matrix shown in FIG. 28A.

The apparatus of FIG. 28A features alternating row flow channels 6048and 6049, intersecting with alternating column flow channels 6050 and6052, with alternative pairs of successive flow channels 6050 and 6052linked by branch flow channels 6086 to define reactor sites at the flowchannel vertices. Each of flow channel row 6048 includes a common inletport 6048 a and an outlet port 6048 b. Each of flow channel rows 6049includes a common inlet port 6049 a and an outlet port 6049 b. Asdescribed below, in certain applications relating to the amplificationof nucleic acids, flow channel rows 6048 may contain a primer, whileflow channel rows 6049 may contain a no-primer control.

Each of alternate flow channel columns 6050 include an inlet port 6050 aand an outlet port 6050 b. Alternate flow channel columns 6052 include acommon inlet port 6052 a and an outlet port 6052 b.

Each reactor 6002 of matrix 6000 is in pressure communication withcontrol lines present in underlying and overlying elastomer layers.Specifically, First control lines 6016 overlap column flow channels 6050and 6052, thereby defining valves 6016 a. First control lines 6016 arein fluid communication with common port 6016 a. Second control lines6035 overlap row flow channels 6048 and branch flow channels 6086,thereby defining valves 6036 and 6038 having different widths. Secondcontrol lines 6035 are in fluid communication with common port 6035 a.

First pump line 6018 underlies column flow channel 6052, therebydefining first push-up pumps 6019. Various separate groupings of firstpump line 6018 are in fluid communication with common ports 6018 a.Second pump line 6022 underlies branch flow channel 6086, therebydefining second push-up pumps 6024. Various groupings of second pumpline 6022 are in fluid communication with common ports 6022 a.

FIG. 28B shows an enlarged plan view of a single reactor 6002 featuringrelatively large control lines 6016 including stabilizing poststructures 6088, column flow line 6050 containing a first templatesample 6008, a column flow line 6052 containing DNA polymerase 6010, anda row flow line 6049 containing primer 6012 or a no-primer control. Therespective volume ratios areV_(template):V_(polymerase):V_(primers)=3:3:4. Rotary pumping valves6019 and 6024 are defined between reactor 6006 and underlying pumpingcontrol channels 6018 and 6022.

FIG. 28C shows a simplified cross-sectional view of the microfluidicmatrix of FIG. 28B taken along line 28C-28C′. FIG. 28D shows asimplified cross-sectional view of the microfluidic matrix of FIG. 28Dtaken along line 28D-28D′.

As shown in FIGS. 28C-D, the device 6000 comprises three layers 6060,6062, and 6064 with distinct functions. Layer 6062 containing matrixflow channel structure 6006 is sandwiched between two layers 6060 and6064 containing integrated hydraulic valves 6016 a and pneumatic pumps6019 and 6024, respectively.

In the middle fluidic layer 6062, flow channels 6006 are 106 pm wide and12˜14 μm high. Each vertex of the matrix 6000 contains a ring shapedchannel (reactor) 6002 of approximate 3 nanoliters. Each row of reactors6002 is connected to a separate input hole 6048 a or 6049 a (about 625μm in diameter) through which unique primers or other reagents may beloaded. Each column can similarly load the reactors with different DNAtemplates or other reagents through second input holes 6050 a. A third,single common input 6052 a for the addition of polymerase or anotherreagent is connected to all the reactors in the matrix.

In the top, control layer 6060, the valve system is designed to loadeach reactor with the three separate reagents while preventing crosscontamination. In total, 2860 valves displayed horizontally orvertically are controlled by only two independent pressure supplythrough-holes 6016 b and 6035 b. Furthermore, the large valves 6038 (270μm wide) or the small valves 6036 (96 μm wide) can selectively beactuated because they have a different threshold of the hydraulicpressure. This is described by Unger et al. in Science, 288, 113-116(2000) and Thorsen et al. in Science, 298, 580-584 (2002). As describedby Unger et al., reagent loading is not blocked by the narrow controlchannels 6035 a (42 μm wide) connecting the valve system, because theirtiny membrane does not deflect at the actuation pressure used.

The third, bottom layer 6064 uses a 20×20 array of rotary pumps in orderto facilitate mixing the reagents. The use of such devices is describedby Chou et al., Biomedical Microdevices, 3, 323 (2001), and by Liu etal., Electrophoresis, 23, 1531-1536 (2002). In the specific embodimentillustrated in FIGS. 28A-D, each reactor 6002 features only two rotarypump structures 6019 and 6024. This represents a simplification overalternative embodiments of microfluidic matrix structures in accordancewith the present invention, such as the alternative embodiment discussedbelow in conjunction with FIGS. 29A-D, wherein three such pumpingstructures underlie each rotary flow channel reactor. A minimum of onlytwo such pumping structures are required to achieve peristaltic pumping,owing to the closed-loop nature of the rotary flow channel reactor atthe vertices.

FIGS. 28E-H show enlarged and simplified plan views of a group of fouradjacent reactors in the matrix of FIGS. 28A-D, illustrating successivesteps of operation of the microfluidic matrix device.

As shown in FIGS. 28E-H, active valves in the top control structure 6060facilitate the loading and isolation of reagents 6008, 6010, and 6012.Specifically, as shown in FIG. 28E, with valves 6016 a actuated, primersets or other reagents 6012 are loaded along rows 6050 and 6052respectively, of the matrix. Actuation of valves 6036 and 6038 isolatesa well-defined volume of primers or other reagents 6012 in each reactor6002.

As shown in FIG. 28F, valves 6036 and 6038 are closed and valves 6016 aopened, to allow for the loading of DNA templates or other reagents 6008down alternate columns 6050 while polymerase or another reagent 6010 issimultaneously introduced to all reactors from a single inlet 6052 athrough alternate columns 6052. Valves 6016 a are then once againactuated, defining the desired volumes of the different reagents 6008and 6010 such as polymerase and templates, and isolating each reactor6006 a.

As described by Unger et al., the different membrane areas of valves6036 and 6038 allow for the selective opening of only valves 6036 byreducing the actuation pressure from 260 kPa to 110 kPa, thus bringingall three components 6008, 6010, and 6012 into fluidic contact withineach reactor 6002. As shown in FIG. 28G, peristaltic pumps 6019 and 6024in the bottom layer allow for the rapid rotary mixing of all reagentswithin the reactors 6002.

FIGS. 29A-D show a set of optical micrographs illustrating the isolationand mixing of different dye solutions within an alternative embodimentof a microfluidic matrix device in accordance with the presentinvention. Four reactors are imaged. The scale bar of FIGS. 29A-D is 600pm.

In FIG. 29A, the “dead-end” control channels are filled in with thegreen dye solution. This is feasible because PDMS is gas-permeable. SeeHansen et al., Proc. Natl. Acad. Sci. U.S.A., 99, 16531-16536 (2002),incorporated by reference herein for all purposes.

In FIG. 29B, different dye solutions are isolated into volume-definedcompartments of the individual reactor. In FIG. 29C, the parabolicprofile 6100 of the Poisueille flow stretches the interfaces of thereagents along the loop channel, producing long and thin interlacedsteams which quickly mix by diffusion (shown here with food dyes), asdescribed by Chou et al. Because of the valve ordering, the solutionsare moving counter-clockwise in the first (top) row of reactors, whileclockwise in the second (bottom) row.

As shown in FIG. 29D, the color-mixed solution results from the rotarypumping for half a minute. A video of the loading and mixing sequencewith food dye is available at http://thebigone.caltech.edu/quake/matrix.

The alternative embodiment of the microfluidic matrix device shown inFIGS. 29A-D differs in certain respects from the embodiment shown FIGS.28A-H. First, as already described, circulation within the reactor isaccomplished with three, rather than two, pumping structures. Owing,however, to the closed loop nature of the isolated reactor, it wasrecognized that peristaltic pumping could be accomplished utilizing onlytwo pumps, thereby simplifying the layout.

In addition, in the embodiment of FIGS. 29A-D, pumps of adjacentreactors are connected by common, serpentine pumping control channels.However, it was observed that the number of reactors which could bepumped by such common pumping lines was limited, owing to accumulateddelay in membrane actuation. Accordingly, the embodiment of FIGS. 28A-Hfeatures pumping control lines organized in groups sharing differentcommon inlets/outlets, thereby reducing accumulated membrane delay.

The protocols of fabricating two-layer polydimethylsiloxane (PDMS)devices using multilayer soft lithography (MSL) have been previouslydescribed, at least by Fu et al., and Hansen et al. For the three-layermicrofluidic matrix elastomeric devices in accordance with embodimentsof the present invention, three separate molds for the different layerswere prepared with UV-lithography. Photoresist of each mold layer may beheated to promote reflow and corresponding formation of roundedfeatures.

The photoresist (Shipley SJR5740) line height was 12-44 μm on thefluidic layer mold or the pump layer mold. A 25 μm thick elastomericlayer with a 20:1 ratio was spin-coated on the fluidic layer mold at1600 RPM for 65 seconds, then cured at 80° C. for 35 minutes.

The mold for the control layer had higher lines of 25 μm, which preventscollapse of the thin elastomeric membrane of the fluidic layer. Tofabricate the control layer, General Electric RTV 615 A and B componentswere mixed in a 4:1 ratio using a Keyence Hybrid mixer. Then the mixturewas poured onto the control layer mold in an foil-coated petri dish andbaked in the oven at 80° C. for 30 minutes to obtain a thick PDMS block(˜3-4 mm).

After incubation, the control layer was peeled from the mold and punchedwith through-holes for pressure connection. It was aligned on the thinfluidic layer and then baked at 80° C. for 45 minutes.

A third pump layer was spin-coated with the RTV615 mixture (20:1) at2200 RPM for 65 seconds, and then incubated at 80° C. for 45 minutes.Next, the bonded device containing the former two control and fluidiclayers was peeled and through-holes were punched for loading fluidicsamples.

The bonded device was then aligned onto the third pump layer and bakedat 80° C. overnight. Then the rest through-holes were punched on thedevice for air injection in pumping. After that, the three-layer devicewas sealed with a piece of glass coverslip (#1, Lakeside microscopeaccessories) and incubated at 80° C. for no less than 3 hours.

III. PCR

Nucleic acid amplification reactions have emerged as powerful tools in avariety of genetic analyses and diagnostic applications. The value ofthese techniques is their ability to rapidly increase the concentrationof target nucleic acids of interest that might be present at very lowand otherwise undetectable levels. For instance, by utilizing thepolymerase chain reaction (PCR) amplification technique, one can amplifya single molecule of a target nucleic acid by 10⁶ to 10⁹.

PCR is perhaps the most well-known of a number of differentamplification techniques. This well established procedure involves therepetition of heating (denaturation) and cooling (annealing) cycles inthe presence of a target nucleic acid, primers that hybridize to thetarget, deoxynucleotides, a polymerase and cofactors such as metal ions.Each cycle produces a doubling of the amount of the target DNA. Thecycles are conducted at characteristic temperatures: 95° C. fordenaturing double stranded nucleic acid, 50 to 65° C. for hybridizationof primer to the target nucleic acid, and 72 to 77° C. for primerextension (see, generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press (1989),incorporated by reference herein for all purposes. See also for example,U.S. Pat. Nos. 4,683,202 and 4,683,195, both of which are incorporatedby reference herein for all purposes.

Methods for conducting PCR amplifications fall into two general classes.The approach typically utilized is a time domain approach in which theamplification reaction mixture is kept stationary and the temperature iscycled. See, e.g., Cheng, et al., Nucleic Acids Res. 24:380-385 (1996);Shoffer, et al., Nucleic Acids Res. 24:375-379 (1996); and Hong, et al.Electrophoresis 22:328-333 (2001), each of which is incorporated hereinby reference for all purposes. While methods utilizing this approach canbe conducted with relatively small sample volumes, the methods requirecomplex regulation of heater elements and relatively long reactiontimes.

Another approach that has been discussed is limited to a space domainapproach in which three temperature zones are constantly kept at thedifferent temperatures and the reaction mixture runs in a serpentineflow channel above it. See, e.g., Kopp et al., Science 280:1046-1048(1998), incorporated by reference herein for all purposes. A method suchas this can be conducted at relatively high speed because it is notnecessary to heat and cool the heaters, but requires the use ofrelatively large sample volumes.

FIG. 34 shows a schematic diagram of one embodiment of a microfluidicstructure 6300 for performing PCR. Top layer 6302 includes controlchannels 6304 a-d for controlling the pumps and valves. Middle layer6304 defines the inlet 6305 a, rotary 6305 b, and outlet 6305 c fluidflow channels. Bottom layer 6306 includes integrated heater structures6308 and electrical leads 6310 in electrical communication therewith.

The loop in the fluid layer forms a rotary pump, by which the PCRreagents can be transported over regions of different temperatures. Thetemperatures are set by tungsten heaters evaporated onto glass, whichbecome the bottom substrate of the nanofluidic chip. The total volume ofthe PCR reaction was 12 nL.

FIG. 35 shows results of Taqman PCR assay performed in the rotary pumpchip. Fluorescence was measured in situ at various time points as thePCR reaction mixture was pumped through different temperature regions. Afragment of the β-actin gene was amplified from human male genomic DNA.The closed circles represent data from an experiment which contained thehuman template DNA. The open circles represent data from a negativecontrol experiment in which template DNA was withheld.

Amplification of purified nucleic acid samples in accordance withembodiments of the present invention is not limited to the specificmicrofluidic structure shown in FIG. 34. Alternative, more complexmicrofluidic geometries are possible.

For example, the microfluidic matrix device shown in FIGS. 28A-H couldbe utilized to perform PCR. In order to test the performance of themicrofluidic matrix chips just described, a number of experiments wereperformed.

Specifically, each row of reactors was alternatively loaded with primersand no-primer controls. Each column of reactors was then loaded with thecDNA template and no-template controls. The DNA template concentrationwas titrated along the positive control columns in order to testsensitivity.

The polymerase was loaded from the single inlet and amortized over allthe independent reactors. The reactors containing all the necessaryreagent components for PCR produced the positive signals (yellow, amixed color of green and dim red); while those reactors that missed oneor two reagent components showed a color of dim red, which derived fromthe passive reference dye in the buffer solution. The expected image isa checkerboard pattern, and any cross-contamination or leakage betweencompartments will be evident.

In these experiments, a 294-bp segment of the human (3-Actin cDNAfragment (1.8 Kbp, Clontech) was amplified. Forward primer and reverseprimer were the following: 5′-TCA CCC ACA CTG TGC CCA TCT ACG A-3′ and5-′CAG CGG AAC CGC TCA TTG CCA ATG G-3′. An amplification protocolsupplied by Applied Biosystems (http://www.appliedbiosystems.com) wasmodified to include the use of DyNAzyme II (0.1 U/reaction, FinnzymesOY) and additives. See Yang et al., Lab on a Chip, 2, 179-187 (2002).The solutions loaded onto the chip were prepared with one component ofthe template, primers or polymerase, and all other necessary PCRreagents and additives.

To simplify the pipetting steps for the checkerboard experiments, twosets of N/2 inputs/outputs are connected together for primer loading.The volumes loaded were 3 μl primers and no-primer control, 1 μl cDNA orno-template control, and 2 μl Dynazyme. An efficient mixing of thereagents was achieved by actuating the rotary pumps with two pneumaticcontrollers (Fluidigm Inc., So. San Francisco) at 10 Hz for 5 minutes.

After mixing was complete, the matrix chip was transferred onto aflat-bed thermocycler (DNA Engine, MJ Research). All the individualreactors were still isolated by the hydraulic control channels, whichreduced the evaporation of the reagents in the fluidic channels to anegligible level.

The whole chip was thermocycled in the following protocol: 2 minutes at50° C., and 1 minute at 96° C.; then 30 cycles: 20 seconds at 96° C.; 40seconds at 60° C. The PCR conditions were verified with conventionalmethods to show that there are no detectable side products from thenon-hot start condition. After thermocycling, the fluorescence emissionfrom the PCR products in the chip (519 nm and 570 nm) was imaged using amodified DNA array scanner (Applied Precision).

FIG. 30 shows a two-color image of fluorescent emission from a 20×20matrix chip. Yellow indicates a positive signal from the sample. Theno-template or no-primer controls show a dim red produced by the passivereference dye in the buffer. The concentration of the DNA templates isas follows (copies per reactor): C1&C3 (6,100), C5&C7 (3,050), C9&C11(610), C13&C15 (305), C17&C19 (61). A threshold fluorescent ratio isestablished to define the false positive/negative signal. Some reactorsin columns 17&19 do not show the desired positive signals, establishingthe sensitivity of the chip. The scale bar of FIG. 30 is 6.4 mm.

FIG. 31 shows a scanned fluorescent image illustrating use of a 10×10matrix chip in which an alternative reagent format was used. The scalebar of FIG. 31 is 6.4 mm.

Specifically, the matrix chip of FIG. 31 was used to explorecombinations of forward and reverse primers. The three main componentswith all the other necessary PCR reagents and additives were as follows:component I included both the cDNA template and the polymerase;components II contained forward primers while component III containedreverse primers. Components II and III were loaded in the rows andcolumns of the matrix, respectively. Then component I, which containedboth the cDNA template and the polymerase, was loaded into all thereactors from the single input.

Even-numbered rows are loaded with the correct forward primers, whileodd rows are negative controls. Columns 1, 3, 5, 6, 8, and 10 are loadedwith the correct reverse primers, while the remainder columns arenegative controls. Thus, one should only observe successful PCRreactions in those reactors at the vertices of correct forward andreverse primers, as shown. Reactor C6R9 shows a false positive signal,which possibly resulted from the contamination of the forward primer ofthe neighboring reactors in the same column (C6).

In this manner, all possible combinations of forward and reverse primerscan be tested with each other on a common DNA template. This format isparticularly useful for PCR optimization and exon mapping.

The chip layout and sample loading sequence are specifically designed sothat DNA template contamination among independent reactors is onlypossible if the valve system fails. Although there exists in principle achance of slight primer contamination, the absolute numbers are so lowthat such an effect should not expected to affect the outcome of theassay. Possible primer contamination can be further reduced by flushingbuffer solution into the fluidic channels before loading the DNAtemplates or polymerase.

In all, six separate 20×20 matrix chips and 8 separate 10×10 matrixchips were tested for a total of 3,200 reactions. Twenty-four out of1550 positive control reactors produced false negative signals. Ineighteen of these cases, the DNA concentrations were close to thedetection limit. The other six reactors failed, possibly because of dustblocking the channels or other fabrication defects.

Thirty-five out of 1,650 negative controls showed false positivesignals, perhaps due to the reagent leakage. In total, 98% of the 3,200reactors tested produced the expected results.

By titrating the template DNA concentration, the detection limit of thechip was established to be around 60 template copies per reactor. FIG.32 plots fluorescent ratio vs. the initial concentration of the cDNAtemplates. Each symbol represents the averaged ratio of 20 reactorscontaining the same template concentration, with an error bar showingthe standard deviation. There is no significant difference between theno-template control and the no-primer control. As the concentrationdecreases to the limit of detection, the standard deviation increasesdue to a mixture of positive and false negative results.

An immediate application of the microfluidic matrix device exists inmedical diagnostics and genetic testing, where the matrix chip allows Npatients to be screened for N mutations or pathogens with PCR or othertechniques such as fluorescent-labeled mismatch-binding protein. SeeBehrensdorf et al., Nucleic Acids Res., 30, e64 (2002).

Another application of interest is gene expression analysis, wherereverse transcriptase PCR can be used to query N mRNA samples for theexpression levels of N different genes.

In another experiment, positive samples were loaded into every reactorin order to collect as much PCR product as possible. The scannedfluorescent image (not shown) demonstrates that all the 400 reactorsworked as expected.

For this experiment, the chip pattern was slightly modified to connecttogether all the inputs/outputs for primer loading/unloading. Thus, thePCR product could be flushed with TE buffer solution (about 40 μl) andcollected with a pipette tip plugged into the wired outputs.

The solution of the extracted PCR amplicon was concentrated into avolume of about 5 μl by evaporation. This pooled amplicon was thenverified by gel electrophoresis. FIG. 33 shows Agarose gelelectrophoresis of the PCR amplicon by the chip or by a microtube. FIG.33 is displayed in 2% Agarose gel with easy-cast electrophoresis system(Model BIA, VWR), stained with Sybr Green I, and documented by the Kodakelectrophoresis system.

The sample of lane 1 of FIG. 33 is 1 μl PCR amplicon by the microtube.The sample of lane 2 of FIG. 33 is 0.5 μl PCR amplicon by the microtube.The sample of lane 3 is a negative control by the microtube. The sampleof lane 4 is 100 bp DNA ladder. The sample of lane 5 is extracted PCRamplicon (400 reactors×3 nl/reactor) by the matrix chip.

Intensity of the pooled amplicon was comparable to the bench-toppositive controls (lane 1 and lane 2). In future devices it should bepossible to integrate on chip capillary electrophoresis for in situproduct analysis beyond the information provided by the Taqman assay.

To summarize, embodiments of microfluidic matrix architectures inaccordance with the present invention represent an effective solution tothe macroscopic/microfluidic interface issue, and demonstrate howmicrofluidics can achieve impressive economies of scale in reducing thecomplexity of pipetting operations. Using an NxN microfluidic matrixwith N=20, N²=400 distinct PCR reactions were performed with only2N+1=41 pipetting steps, compared with the 3N²=1,200 steps required withconventional fluid handling. Each vertex of the matrix has a 3 nanoliterreactor, and a single 2 μL aliquot of polymerase is amortized over all400 independent reactions, thus dramatically reducing sample overheadand minimizing reagent consumption.

While the specific embodiment shown and described above performs PCR bythe introduction of primers, nucleic acid samples, and enzymes throughseparate inlets, this is not required by the present invention. Inaccordance with one alternative embodiment, the three separate inletchannels can be utilized to flow forward primers, reverse primers, and amixture of nucleic acid sample and enzyme, respectively, into the threeinlets to the flow channels of a microfluidic matrix device.

And while the specific embodiment of a microfluidic device shown anddescribed above is fabricated from three layers of elastomer material,this is also not required by the present invention. In accordance withalternative embodiments, control lines of different widths definingmembranes of varying size that are actuable at different appliedpressures, could formed in a single elastomer layer adjacent to thefluidic flow layer. These various control lines could be utilized toperform reactor gating, isolation, and circulation functions.

Moreover, the embodiments of microfluidic devices in accordance with thepresent invention are not limited to the specific matrix architectureshown and described in connection with FIGS. 28A-H and 29A-D. Forexample, alternative embodiments of microfluidic devices in accordancewith the present invention could include features that allow forrecovery of the contents of individual reactors following reaction.FIGS. 36A-D show simplified plan views showing operation of one suchalternative embodiment.

FIG. 36A shows an initial step of operation, wherein reactor 6202 ofmicrofluidic matrix device 6200 has been loaded with reagents throughcolumn flow channel pairs 6204 and 6206, followed by reaction bycirculation within reactor 6202 isolated by actuation of adjacent valves6208, to form reaction product 6218. The pumping structures responsiblefor circulating fluid within the reactor 6202 have been omitted fromFIGS. 36A-D for clarity of illustration.

The architecture of the microfluidic matrix device shown in FIG. 36Adiffers from that of prior Figures in two important respects. First,each rotary reactor 6202 is fabricated adjacent to a holding chamber6210, that is in turn in fluid communication with an additional columnflow channel 6212 gated by valves 6214. As described below in connectionwith FIGS. 36B-C, holding chamber 6210 and recovery flow channel 6212play a key role in permitting recovery of the contents of individualreactors from the microfluidic matrix.

A second key difference between the architecture of the microfluidicmatrix device shown in FIG. 36A and those discussed previously, is thatthe reactors are formed from pairs of horizontal branch channelsconnecting alternative pairs of column flow channels, rather than fromcontinuous row flow channels. This architectural aspect becomesimportant in ensuring efficient and complete transfer of the contents ofthe reactor to the adjacent holding chamber, as shown in FIG. 36B.

Specifically, leaving valves 6208 open, buffer solution is flowed intorow flow channel 6216, thereby displacing reaction product 6218 presentin reactor 6002 into holding chamber 6210. Offsetting the position ofupper branch flow channel 6220 from row flow channel 6216 in thisembodiment, ensures that the contents of the reactor are captured andtransferred during this process.

FIG. 36C shows the next step in the process, wherein the reactionproduct 6218 is isolated in holding chamber 6210, by reactuating valves6208. FIG. 36D shows the final step in the process, wherein the reactionproduct 6218 may be flushed from the microfluidic device and recoveredat an outlet or other feature, due to the positive pressure withincolumn recovery flow channel 6212.

It should be noted that when valves 6208 or 6214 are opened, somecontamination may occur between neighboring reactors. Such contaminationmay be caused by convective mixing between the contents of adjacentreactors during pumping along the common row flow channel as shown inFIG. 36B. However, the contamination can be reduced to a minimum levelby isolating the contents with the neutral buffer solution in theholding chambers.

Contamination may also result from the passive diffusion of materials,for example between adjacent reaction products pumped along a commoncolumn flow channel as shown in FIG. 36D. However, the amount of suchcontamination is slight, and its impact can often be neglected for manyapplications. For example, a reaction product comprising medium or highmolecular weight DNA takes hours to diffuse a distance of hundreds ofmicrons, while the process of recovering the specified product from thechip can be accomplished in only a few seconds.

In some instances, the microfluidic devices described herein can be usedas an analytical tool to amplify a target nucleic acid potentiallypresent in a sample and then detect the amplified product to determinewhether the target nucleic acid is present or absent in the sample.Thus, amplification serves to enhance the ability to detect targetnucleic acids present at low levels. When utilized in this manner, thedevices can be used in a wide variety of different applications.

For example, the devices can be used in various diagnostic applicationsthat involve a determination of whether a particular nucleic acid ispresent in a sample. Hence, samples can be tested for the presence of aparticular nucleic acid associated with particular pathogens (e.g.,certain viruses, bacteria or fungi), for instance. The devices can alsobe utilized for identification purposes, such as in paternity andforensic cases.

The methods and devices provided herein can also be utilized to amplifylow levels of nucleic acid for further examination to detect orcharacterize specific nucleic acids that are correlated with infectiousdiseases, genetic disorders or cellular disorders (e.g., oncogenesassociated with cancer). Genetic disorders are those that involve aspecific deletion and/or mutation in genomic DNA. Examples of geneticdiseases that can be detected include, but are not limited to, α- andβ-thalassemia, cystic fibrosis and sickle cell anemia. Because thedevices and methods disclosed herein can utilize very small samplevolumes, they are useful in amplifying DNA samples obtained inconjunction with the prenatal diagnosis of genetic disease.

However, the amplification reactions can also be utilized as just onestep of a more extensive process involving the diagnostic testing forparticular target nucleic acids and in preparing sufficient nucleic acidfor use in various genetic engineering applications. Hence, amplifiedsample can be used in a number of post amplification manipulations andanalyses. Examples of such post amplification processes and analysesinclude, but are not limited to, sequencing of amplified products,cell-typing, DNA fingerprinting and mapping of DNA sequences.

Amplified products can also be generated for use in various geneticengineering applications. For instance, amplified product can beutilized to conduct recombination studies. In other applications, thedevices are used to produce target DNA for insertion into a variety ofvectors. Such vectors can then be used to transform cells for theproduction of desired products such as proteins, or nucleic acids invarious therapeutic or biotechnological processes.

The microfluidic devices in accordance with embodiments of the presentinvention can also be utilized to conduct sequencing reactions such aschain termination methods using dideoxynucleotides. Sequencing reactionsutilizing the devices disclosed herein can be conducted in differentformats. One approach is to conduct four separate sequencing reactions,a separate reaction being conducted in four different thermal cyclingdevices as provided herein. Each of the four reactions contains targetnucleic acid, a primer complementary to the target, a mixture of onedideoxynucleotide (ddNTP) (optionally labeled) with its counterpartdeoxynucleotide (dNTP), and the other three dNTPs. Thus, each one of thereactions is conducted with a different ddNTP/dNTP mix. Followingcompletion of the primer extension reactions, the different sizedextension products can be separated by capillary gel electrophoresis.This separation can be performed in a separation module as describedsupra that is integrated with the present devices or in a stand alonecapillary gel electrophoresis apparatus.

In addition, embodiments of microfluidic matrix devices can also beutilized in more streamlined formats in which reactions are conductedsimultaneously in a single thermal cycling device using differentiallylabeled dideoxynucleotides. The resulting mixture of chain-terminatedreaction products are then separated on a single capillary gelelectrophoresis column. The identity of the dideoxynucleotideincorporated into the primer can be determined on the basis of thelabel.

Restriction digests of nucleic acids can also be conducted utilizing themicrofluidic matrix devices in accordance with embodiments of thepresent invention. Temperature control in such reactions initiallyinvolves controlling the temperature within the device at a temperaturethat promotes the activity of the restriction enzyme (e.g., 1-3 hours at30-50° C. depending upon the particular enzyme. The other temperature isselected to promote enzyme deactivation (e.g., 60° C. for 20 minutes).

Analyses to determine the identity of a nucleotide present at apolymorphic site, i.e. the site of variation between allelic sequencessuch as a single nucleotide polymorphism (SNP), can also be conductedwith certain of the present devices. Often these analyses are conductedusing single base pair extension (SBPE) reaction. A number of SPBEassays have been developed, but the general approach is quite similar.Typically, these assays involve hybridizing a primer that iscomplementary to a target nucleic acid such that the 3′ end of theprimer is immediately 5′ of the variant site or is adjacent thereto.Extension is conducted in the presence of one or more labelednon-extendible nucleotides that are complementary to the nucleotide(s)that occupy the variant site and a polymerase. The non-extendiblenucleotide is a nucleotide analog that prevents further extension by thepolymerase once incorporated into the primer. If the addednon-extendible nucleotide(s) is(are) complementary to the nucleotide atthe variant site, then a labeled non-extendible nucleotide isincorporated onto the 3′ end of the primer to generate a labeledextension product. Hence, extended primers provide an indication ofwhich nucleotide is present at the variant site of a target nucleicacid. Such methods and related methods are discussed, for example, inU.S. Pat. Nos. 5,846,710; 6,004,744; 5,888,819; 5,856,092; and5,710,028; and in WO 92/16657, each of which is incorporated herein byreference for all purposes.

Using devices as described herein, the temperature within a temperaturecontrol region can be selected to promote the primer annealing, primerextension and denaturation steps involved in these particular analyses,and thus allows these extension reactions to be conducted in a thermalcycling format.

IV. Other Applications

Beyond PCR, the matrix chip provides a general method to performchemical and biological experiments with precious reagents in a highlyautomated fashion. For example, while the foregoing discussion of themicrofluidic matrix devices has focused on their utility in conducting alarge number of nucleic acid amplification reactions, it will beappreciated by those with ordinary skill in the art that suchmicrofluidic devices can be utilized to conduct a wide variety of typesof reactions and screening methods. Thus, by way of illustration but notlimitation, the devices can be utilized to conduct synthetic reactionsbetween a plurality of reactants. Using the device shown in FIGS. 28A-H,for instance, a first set of reagents can be introduced into thehorizontal flow channels, a second set of reagents can be introducedinto the vertical flow channels that have an independent inlet; and athird reagent can be introduced into the vertical flow channels that areconnected to the shared inlet. Using the metering and mixing techniquesdiscussed above, these various reagents can be combined within reactorsformed at the vertices of the matrix in order to accomplish reaction.

Embodiments of microfluidic matrix devices according to the presentinvention can also be utilized to screen compounds for a desiredactivity. With the devices described in FIGS. 28A-H and 29A-D forexample, typical screening methods involve introducing a set of testcompounds into the horizontal flow channels, with another set ofcompounds, cells, vesicles or the like being introduced via the verticalflow channels. Mixing occurs at the junctions and the presence orabsence of the desired activity can then be monitored at vertices of thematrix.

For instance, a wide variety of binding assays can be conductedutilizing the microfluidic devices disclosed herein. Interactionsbetween essentially any ligand and antiligand can be detected. Examplesof ligand/antiligand binding interactions that can be investigatedinclude, but are not limited to, enzyme/ligand interactions (e.g.,substrates, cofactors, inhibitors); receptor/ligand; antigen/antibody;protein/protein (homophilic/heterophilic interactions); protein/nucleicacid; DNA/DNA; and DNA/RNA. Thus, the assays can be used to identifyagonists and antagonists to receptors of interest, to identify ligandsable to bind receptors and trigger an intracellular signal cascade, andto identify complementary nucleic acids, for example. Assays can beconducted in direct binding formats in which a ligand and putativeantiligand are contacted with one another or in competitive bindingformats well known to those of ordinary skill in the art. Binding assayscan be conducted in heterogenous formats, as well as homogenous formats.In the homogeneous formats, ligands and antiligands are contacted withone another in solution and binding complexes detected without having toremove uncomplexed ligands and antiligands. Two approaches frequentlyutilized to conduct homogenous assays are fluorescence polarization (FP)and FRET assays.

Immunological assays are one general category of assays that can beperformed with certain of the microfluidic devices disclosed herein.Some assays are conducted to screen a population of antibodies for thosethat can specifically bind to a particular antigen of interest. In suchassays, a test antibody or population of antibodies is contacted withthe antigen. Typically, the antigen is attached to a solid support.Examples of immunological assays include enzyme linked immunosorbentassays (ELISA) and competitive assays as are known in the art.

A variety of enzymatic assays can be performed using some of the devicesdisclosed herein. Such enzymatic assays generally involve introducing anassay mixture containing the necessary components to conduct an assayinto a flow channel or junction for reaction with an enzyme that issubsequently introduced. The assay mixtures typically contain thesubstrate(s) for the enzyme, necessary cofactors (e.g., metal ions,NADH, NAPDH), and buffer, for example. If a coupled assay is to beperformed, the assay solution will also generally contain the enzyme,substrate(s) and cofactors necessary for the enzymatic couple.

A number of different cell reporter assays can be conducted with theprovided microfluidic devices. One common type of reporter assay thatcan be conducted include those designed to identify agents that can bindto a cellular receptor and trigger the activation of an intracellularsignal or signal cascade that activates transcription of a reporterconstruct. Such assays are useful for identifying compounds that canactivate expression of a gene of interest. Two-hybrid assays, discussedbelow, are another major group of cell reporter assays that can beperformed with the devices. The two-hybrid assays are useful forinvestigating binding interactions between proteins.

Cells utilized in screening compounds to identify those able to triggergene expression typically express a receptor of interest and harbor aheterologous reporter construct. The receptor is one which activatestranscription of a gene upon binding of a ligand to the receptor. Thereporter construct is usually a vector that includes a transcriptionalcontrol element and a reporter gene operably linked thereto. Thetranscriptional control element is a genetic element that is responsiveto an intracellular signal (e.g., a transcription factor) generated uponbinding of a ligand to the receptor under investigation. The reportergene encodes a detectable transcriptional or translational product.Often the reporter (e.g., an enzyme) can generate an optical signal thatcan be detected by a detector associated with a microfluidic device.

In addition to the assays just described, a variety of methods to assayfor cell membrane potential can be conducted with the microfluidicdevices disclosed herein. In general, methods for monitoring membranepotential and ion channel activity can be measured using two alternatemethods. One general approach is to use fluorescent ion shelters tomeasure bulk changes in ion concentrations inside cells. The secondgeneral approach is to use of FRET dyes sensitive to membrane potential.

Assays of cell proliferation can also be monitored with certain of themicrofluidic devices disclosed herein. Such assays can be utilized in avariety of different studies. For example, the cell proliferation assayscan be utilized in toxicological analyses, for example. Cellproliferation assays also have value in screening compounds for thetreatment of various cell proliferation disorders including tumors.

The microfluidic devices disclosed herein can be utilized to perform avariety of different assays designed to identify toxic conditions,screen agents for potential toxicity, investigate cellular responses totoxic insults and assay for cell death. A variety of differentparameters can be monitored to assess toxicity. Examples of suchparameters include, but are not limited to, cell proliferation,monitoring activation of cellular pathways for toxicological responsesby gene or protein expression analysis, DNA fragmentation; changes inthe composition of cellular membranes, membrane permeability, activationof components of death-receptors or downstream signaling pathways (e.g.,caspases), generic stress responses, NF-kappaB activation and responsesto mitogens. Related assays are used to assay for apoptosis (aprogrammed process of cell death) and necrosis.

By contacting various microbial cells with different test compounds,some of the devices provided herein can be used to conduct antimicrobialassays, thereby identifying potential antibacterial compounds. The term“microbe” as used herein refers to any microscopic and/or unicellularfungus, any bacteria or any protozoan. Some antimicrobial assays involveretaining a cell in a cell cage and contacting it with at least onepotential antimicrobial compound. The effect of the compound can bedetected as any detectable change in the health and/or metabolism of thecell. Examples of such changes, include but are not limited to,alteration in growth, cell proliferation, cell differentiation, geneexpression, cell division and the like.

Additional discussion of biological assays that can be conducted withcertain of the microfluidic devices disclosed herein is provided incommonly owned PCT application PCT/US01/44869, filed Nov. 16, 2001.

Beyond uses in performing multiple independent assays, embodiments ofmicrofluidic matrix devices in accordance with the present inventioncould also be used for multi-step assays. For example, after thereactions of the first-step are completed in the formats demonstratedabove, the products may be isolated into the central compartments ofeach of the individual reactors. The other compartments may then befilled with the reagents for the downstream assays by operating thevalve systems exactly in the same way as before. Therefore, theintermediate products need not be extracted off the chip, facilitatingthe automation of the multi-step assays and simplifying chip design andoperation. Furthermore, this approach can save the unavoidable loss ordilution of product in the dead volume of the chip and the outlet port.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention, but that the invention will include all embodiments andequivalents falling within the scope of the claims.

1. A microfluidic device comprising: a plurality of flow channelsdefined within a first layer to form a matrix of rotary flow reactors; afirst set of control lines proximate to and separated from the flowchannels by first membranes, the first membranes actuable to introducefluids into the rotary flow reactors and to isolate the rotary flowreactors, and wherein the first set of control lines are defined in asecond layer adjacent to the first layer; and a second set of controllines proximate to and separated from the flow channels by secondmembranes, the second membranes actuable to cause peristaltic pumping ofthe fluids within the isolated rotary flow reactors, and wherein thesecond set of control lines are defined in a third layer adjacent to thefirst layer on a side opposite to the second layer.
 2. The microfluidicdevice of claim 1 wherein the second layer overlies the first layer, andthe third layer underlies the first layer.
 3. The microfluidic device ofclaim 1 wherein the second layer underlies the first layer, and thethird layer overlies the first layer.
 4. The micro fluidic device ofclaim 1 wherein the flow channels are oriented in rows and columns, andthe reactors are formed by intersection of a selection from a groupconsisting of: a row flow channel and a column flow channel; alternatesuccessive pairs of a row flow channel or a column flow channel; and abranch channel connecting the alternate successive pairs of the row orthe column flow channels.
 5. The microfluidic device of claim 1 whereinthe flow channels are oriented in rows and columns, and the reactors areformed by branch flow channels connecting alternate successive pairs ofone of row and column flow channels.
 6. The microfluidic device of claim1 wherein the first set of control lines exhibit a first width and asecond width greater than the first width, a first membrane portiondefined by the first width and actuable by a greater applied pressure toisolate the reactor, and a second membrane portion defined by the secondwidth and actuable by a lesser applied pressure to permit circulation offluid within the reactor.
 7. The microfluidic device of claim 1 whereinthe first set of control lines contain water and the second set ofcontrol lines contain air.
 8. The microfluidic device of claim 1 whereinthe second set of control lines are organized in groups sharingdifferent common inlets and outlets.
 9. The microfluidic device of claim1 wherein the second set of control lines share common inlets andoutlets.
 10. The microfluidic device of claim 1 further comprising: aholding chamber in fluidic communication with a reactor; and a recoveryflow channel in fluidic communication with the holding chamber andconfigured to receive contents of the reactor.
 11. The microfluidicdevice of claim 10 wherein: the flow channels are oriented in rows andcolumns; the reactor is formed by a branch flow channel connecting analternate successive pair of one of row and column flow channels; theholding chamber is in fluidic communication with the reactor along theother of the row and column flow channels; and the recovery flow channelis parallel to the other of the row and column flow channels.
 12. Amethod of conducting a chemical reaction, the method comprising:providing a microfluidic device comprising; a plurality of flow channelsdefined within a first layer to intersect and form a matrix of rotaryflow reactors; a first set of control lines proximate to and separatedfrom the flow channels by first membranes, wherein the first set ofcontrol lines are defined in a second layer adjacent to the first layer;a second set of control lines proximate to and separated from the flowchannels by second membranes, wherein the second set of control linesare defined in a third layer adjacent to the first layer on a sideopposite to the second layer; introducing first and second chemicals tothe rotary flow reactors; applying pressure to the first set of controllines to actuate the first membranes to isolate the rotary flowreactors; and applying pressure to the second set of control lines toactuate the second membranes to cause peristaltic pumping of the fluidswithin the isolated rotary flow reactors.
 13. The method of claim 12wherein the flow channels are oriented in rows and columns, and thereactors are formed by branch flow channels connecting alternatesuccessive pairs of one of rows and columns, the method comprising:flowing the first chemical to the reactor through a row flow channel;and flowing the second chemical to the reactor through a column flowchannel.
 14. The method of claim 13 wherein the alternate successivepairs of one of the rows and columns have separate inlets, the methodfurther comprising flowing a third chemical to the reactor through theone of the alternate row and column flow channels.
 15. The method ofclaim 14 wherein flowing the first chemical comprises flowing a primer,flowing the second chemical comprises flowing a nucleic acid sample, andflowing the third chemical comprises flowing an enzyme.
 16. The methodof claim 15 further comprising heating the microfluidic device toamplify the nucleic acid sample.
 17. The method of claim 14 whereinflowing the first chemical comprises flowing a nucleic acid sample,flowing the second chemical comprises flowing a primer, and flowing thethird chemical comprises flowing an enzyme.
 18. The method of claim 17further comprising heating the microfluidic device to amplify thenucleic acid sample.
 19. The method of claim 14 wherein flowing thefirst chemical comprises flowing a forward primer, flowing the secondchemical comprises flowing a reverse primer, and flowing the thirdchemical comprises flowing a mixture of an enzyme and a nucleic acidsample.
 20. The method of claim 19 further comprising heating themicrofluidic device to amplify the nucleic acid sample.
 21. The methodof claim 12 wherein: applying pressure to the first set of control linescomprises applying pressure to a water-filled control line; and applyingpressure to the second set of control lines comprises applying pressureto an air-filled control line.
 22. The method of claim 12 whereinapplying pressure to the second set of control lines comprises applyingpressure to a plurality of inputs.
 23. The method of claim 12 furthercomprising recovering a contents of the rotary flow reactors after theperistaltic pumping.
 24. The method of claim 23 wherein recovering thecontents comprises: flowing the contents from the reactor into anadjacent holding chamber; and flowing the contents from the holdingchamber along a recovery flow channel to an outlet.
 25. The method ofclaim 24 wherein the contents are flowed to the outlet orthogonal to adirection of flow of the contents to the holding chamber.